The ability of pathogens to acquire iron from the iron-deficient mammalian environment is one important parameter in the outcome of the infectious disease process. Although virulence studies with Yersinia pestis were one of the first indications that iron availability influenced the outcome of bacterial infections, the iron acquisition systems of Y. pestis are still ill-defined. The pigmentation (Pgm+) phenotype of Y. pestis originally referred to the ability of wild-type cells, grown at 26 degrees C, to adsorb large quantities of exogenous hemin but now includes other traits, such as expression of several iron-regulated proteins, 37 degrees C growth in iron-chelated medium, and sensitivity to the bacteriocin, pesticin, that may be either directly or indirectly involved in iron metabolism. Spontaneous Pgm- mutants lose all the above characteristics and are avirulent. Thus, expression of iron-regulated genes and a transport system are linked to the pgm locus which is essential to the virulence of the plague bacillus. The specific aims of this proposal are to 1) characterize Pgm-linked, iron-regulated genes; 2) examine a newly identified Pgm- and siderophore-independent iron uptake system (Yfe); 3) analyze regulatory mechanisms controlling expression of iron-regulated genes; and 4) characterize the physiological roles and virulence properties of these iron-regulated proteins. Insertional mutagenesis, DNA sequencing, and reporter gene fusions, will be used to define the genetic organization and regulation of iron-regulated genes. Protein analyses and iron utilization studies will determine their biochemical features and possible physiological roles. Engineered mutants will determine the roles of these systems in survival and growth in fleas, mammals, and phagocytic cells. The long-term objec- tives of this research are to determine the importance of iron uptake systems in different in vivo environments to the pathogenesis of bubonic plague. Y. pestis represents a model which relies exclusively on siderophore-independent iron transport systems, which are ill-defined in all pathogens that possess such systems. An understanding of the compositions and functions of these systems and their pathogenic roles may lead to their use as protective antigens or as targets for new drugs. These results will also provide general insights into siderophore-independent iron uptake systems, their regulation, and importance in the infectious disease process that may be relevant to other highly virulent facultative intracellular parasites.